Process and means for down stream processing

ABSTRACT

A method for recovering a desired component from a sample solution with beads under stabilized fluidized bed conditions is provided. Also provided are support beads for use in downstream processing which comprise a polymer matrix into which glass or silica particles have been incorporated.

[0001] The present invention is related to the field of separations influidized beds and to beads of a polymer, especially a polysaccharide,into which glass or silica particles, especially quartz particles havebeen incorporated, and the use of these beads as carrier matrices instabilized fluidized bed systems, which are characterized by having alow extent of axial dispersion.

[0002] It is known that solid entities may be kept suspended above asolid support by bringing the solid entities into a fluid medium (gas orliquid) which is flowing in opposite direction relative to thegravitational field. A number of particles placed in such a stream offlowing medium in a confined space, such as a cylindrical vessel(column), is commonly referred to as a fluidized bed provided that theparticles stay resident in the confined space. This is achieved bybalancing the gravitational force versus the frictional, lifting force,exerted by the fluid stream on the solid particles.

[0003] Fluidized beds have been used as an efficient means of bringingsolid particles in contact with a fluid phase. The relatively high fluidvelocities, relative to the particles, allow for efficient mass and heattransfer. Conseguently, fluidized beds have been used for combustion andfor adsorption processes. Fluidized beds have also been used forculturing of microbial plant or animal cells in so called air-liftreactors. In this case nutritients and dissolved oxygen are brought tothe cells and waste products are removed from the cells efficientlybecause of the efficient mass transfer.

[0004] Minor irregularities in the flow field in a fluidized bed causetranslational movements of the particles. Over a certain time, there isthe same probability that a certain particle may be found at anyposition within the confined space of the fluidized bed. Here, thiseffect is referred to as back-mixing or a large degree of axialdispersion. The back-mixing is advantageous when it is desirable toachieve a homogeneous composition of the fluid and solid phases in theentire fluidized bed. However, for adsorption processes, a homogeneouscomposition is not necessarily advantageous.

[0005] In fact, it is possible to obtain a lower concentration of asolute in the effluent fluid if there is no back-mixing than there iswith back-mixing in the confined space of the fluidized bed.

[0006] In order to prevent complete back-mixing, it has been shown(Buijs (1980)) that screens inserted into the fluidized bed result in acompartmentalization of the bed.

[0007] The density, viscosity and the velocity of the fluidium and thediameter and density of the solid entities affect the balancing offrictional versus gravitational forces (Lydersen (1979)). Much lowerfluid velocities must be used with liquids than with gases because ofthe higher densities and viscosities of liquids. In order to reduce masstransfer resistances and to increase through-put (the liquid feed-rate)one may wish to use higher flow rates than those which may be balancedby the gravitational field only. This is possible by applying a thirdforce onto the solid entities. The latter force may be induced by amagnetic field applied to a bed containing ferro- or paramagneticparticles. Such magnetically stabilized fluidized beds have beendescribed (see for instance Burns (1985:1 and 1985:2)). The heatgenerated by such a system for stabilizing the bed is a cleardisadvantage and makes the method less useful especially in temperaturesensitive biomolecular systems, even if various cooling systems areavailable. The need for equipments for generating a magnetic field andfor cooling the system increases the process costs considerably.

[0008] Particles with relatively lower density and diameter move upwardsin the fluidized bed. Consequently, it is possible to feed anunclarified liquid into a fluidized bed containing large diameter and/orhigh density adsorbent particles without accumulation of solids from thefeed in the fluidized bed. The relatively smaller and/or less denseparticles originating from the feed stream will be washed out with theeffluent provided that the liquid flow-rate is chosen properly. Thus itis possible to improve an adsorptive recovery process starting with anunclarified feed by using a fluidized bed since time, costs and yieldreduction caused by a clarification step are avoided. However, since afluidized bed is back-mixed (unless it is stabilized in some way) thebed is a less efficient adsorber than a packed bed due to the largeraxial dispersion in the former. A second reason why the packed bed ismore efficient as an adsorber is that the packed bed acts as amulti-step adsorber.

[0009] A single step adsorber, such as a fluidized bed, may, ontheoretical grounds, be expected to work efficiently if the adsorbingsites have a high binding affinity for a solute, relative to theconcentration of the solute in the feed stream. The systems described sofar for carrying out separations in a fluidized bed, however, do notfulfil all the demands raised by the users. One problem so far has beenthat since the beds are not stabilized, flow channels through the bedare easily created, in which the sample molecule might pass with onlylittle chance to be bound.

[0010] The beads should be relatively small to allow short diffusiondistances, have high density, controlled high sedimentation velocity,being an easily derivatized hydrophilic polymer suitable forapplications involving biomolecules.

[0011] We have now unexpectedly found that beds may be designed, whichcombine the advantageous properties of packed beds and fluidized beds.In one aspect of the invention beds are provided which allow relativelyless dense and/or relatively smaller particles to pass through the bedwith the upward flowing liquid stream since the particles are not packedclose together.

[0012] By using bead particles covering a given size and/or densityintegral the particles are kept from moving around in the confined spaceof the bed such that( for a certain particle the Probability to find itin a certain position is high only in a limited volume being a minutefraction of the total bed volume. By keeping the particles residentlocally, back-mixing is prevented, thereby reducing axial dispersion andallowing for multistep adsorption without insertion of screens orsimilar devices.

[0013] Furthermore, the beads of the invention are kept suspended inupward flowing liquids without the need for application of heatgenerating magnetic fields or use of ferro- or paramagnetic additives tothe adsorbent particles. In the following the beds achieved when usingthe actual particles are referred to as stabilized or unmixed expandedbeds, which are characterized by having negligible axial dispersion. Theaxial dispersion is often expressed by the vessel dispersion number (fora definition see Levenspiel (1972)) which in a stabilized bed should beless than about 75×10⁻³, and especially less than 20×10⁻³.

[0014] The beads according to the invention comprises a polymer matrixinto which glass or silica particles, preferably quartz particles areincorporated. The beads may be porous as well as non-porous. Thediameter is 100-1000 μm, preferably 100-500 μm, and spherical beads aswell beads of an irregular shape may be used, even if spherical beadsare preferred as discussed below. The density of the beads is typically1.10-1.50 g/ml, for instance around 1.15 g/ml (the values are given forhydrated beads).

[0015] The polymer is synthetic, for instance from mono- or polyvinylmonomers like acrylates, metacrylates or vinylbenzenes, or of naturalorigin, preferably a polysaccharide, for instance agarose, starch,cellulose or derivatives of these, optionally crosslinked for thedesired rigidity and pore distribution.

[0016] The glass or silica particles to be incorporated are preferablyin the range of from 1-100 μm and ray be spherical as well as of anirregular shame. The amount of silica particles incorporated into thepolymer particles is in the range of from 5-50% of the weight of the setfinal particle. In a preferred embodiment of the invention quartzparticles are utilized.

[0017] One important characteristic of a carrier matrix to be used indown stream processing is that it is stable to the various solutionsused for instance in reconditioning steps. Several applications requirea strongly alkaline medium to be used for reconditioning and this hasbeen found to be fatal to the prior art carrier matrices earlier tested.Especially in cases when the flow through the bed during the elutionstep is reversed to give a packed bed, even minor changes of the supportmatrix might cause serious problems resulting in lower yields, etc. Inthis elution technique spherical beads are preferred for improvedelution capability. The reasons for this are well known to those skilledin the art.

[0018] In this aspect of the invention the beads are used in aseparation system of the type schematically shown in FIG. 1, especiallyadopted for recovery of products from a culture medium, including forinstance cell wall particles and other waste products after lysis of thebacteria or other hosts that have been used.

[0019] The major components of the system are: pumps P1-P3 (supplyingthe appropriate solutions), valves V1-V3, sample container S, column C,containing a flow distributor D at the bottom and an adjustable upperadapter (not shown), and a fraction collector FC.

[0020]FIG. 1a shows the system in the fluidized bed mode during whichthe desired component is bound to the gel and separated from othercomponents. A solution is pumped by P2 into the column, containing thebeads, which have been derivatized to bind the component to be purified,for creating a stabilized fluidized bed and for creating appropriateconditions with regard to pH, ionic strength, etc. The outflow from thecolumn is connected to waste. The valve V1 is next switched so that thesample solution is pumped by P1 into the column, under conditions sothat a stabilized fluidized bed is maintained. When she sample has beentransferred to the column, valve V1 is switched so that a washingsolution (via P2) is fed into the column. After the fluidized bed hasbeen thoroughly washed, and unbound components like cell particles etchave been eluted to waste through the top of the column, the first phaseof the procedure is finished.

[0021] In FIG. 1b the flow through the column is reversed for elution ofthe bound sample components. The valves have been switched so that theflow is from P3 via the column to the fraction collector FC. After thebed has been allowed to settle, and optionally further converted to apacked bed by moving the upper adapter down to the bed surface, asuitable elution solution is via P3 introduced into the bed whereby thedesired component is released and collected. The upper adapter, which isof the traditional type found in a great number of columns forchromatography, is adjustable to a desired position in the column andcontains a net to prevent beads to pass.

[0022] In an alternative embodiment of the invention elution of thecolumn is carried out in the fluidized mode by introducing the elutionsolution via pump P2 into the column and collecting the sampleconstituents released from the beads with a fraction collector connectedto the upper outlet of the column (replacing waste in FIG. 1a). In thiscase there is no need for a movable upper adapter in the column.

[0023] Of crucial importance for obtaining the stabilized beds of theinvention is the way the solution is introduced into the column. Thesolution must be equally distributed over the cross sectional area ofthe column and in order to achieve this a distributor is utilized. Thedistributor could typically be a perforated plate on the upper side (thegel bed side). It need not to be mentioned that the holes in the platemust be great enough to allow all sample constituents to pass orotherwise clogging would disturb the procedure. The pressure drop over Dmust for each combination of flow velocity, viscosity, particle sizeetc, exceed a given value since otherwise flow channels with low flowresistance and accordingly high flow are created in the bed. The ratiobetween the pressure drop over the distributor and the bed shouldtherefore exceed 3% and preferably 10%, with much. higher values, suchas up to 50%, 500% and even 2000% being typical in an optimized system.The total cross sectional area of the holes is typically about 0.005 to10%, especially 0.005 to 3%, of the total area of the plate. If aconical distributor is used the need for a certain pressure drop in thedistributor is less pronounced for obvious geometrical reasons andvalues below those indicated above are accessible.

[0024] The dimensions of a column to be used in a process according tothe invention cover a broad range of values, for instance a diameterfrom 1 cm up to 1-2 meters and a length of about 10 cm to 10 meter,depending on the type of separation to be carried out and on what scaleit is to be done. An important finding is that considerably shortercolumns can be used in the stabilized fluid bed process compared to thenon-stabilized prior art systems. The process is accessible for flowvelocities around at least 50-3000 cm/h, even if values of about 100-500cm/h are most frequently used. During elution the flow is normally5-500cm/h, and in particular 50-200 cm/h.

[0025] The method can as mentioned above be used in systems containingalso solid particles like precipitates and cell wall constituents fromcell culture media. In traditional chromatographic systems this type ofimpurities requires one or more prepurification step. This isaccordingly one of the major advantages of the claimed method in thatbacteria or yeast homogenates as well as blood, urine etc from humans oranimals can be introduced directly into the column.

[0026] It has been found that in systems containing considerable amountsof particulate impurities clogging might occur in the upper adapter.This can easily be avoided if the flow through the column isoccasionally reversed during the adsorption phase. An intermittent flowreversal of about 10-20 seconds every 15th minute has been found toeliminate the problem.

[0027] In a typical example a culture medium with a pH value of about 7and containing immunoglobulins is fed as illustrated in FIG. 1a to acolumn containing beads derivatized with protein A which will bind IgG.Waste components, including particles, are washed away with a neutralbuffer, the flow is reversed and when the packed bed has been formed,the pH of the elution buffer is decreased to about 3 and IgG is elutedthrough the bottom of the column, in the mode illustrated in FIG 1 b.

[0028] The invention is accordingly in one aspect related to supportbeads for use in down stream processing, such as fluidized bedseparations, comprising a polymer matrix, into which silica particleshave been incorporated. The polymer is preferably a polysaccharide, forinstance agarose, starch, cellulose or derivatives of these, optionallycrosslinked for the desired rigidity and pore distribution. The beadsare in the range of from 100-1000 μm, preferably 100-500 μm and withglass or silica incorporated into the polymer particle in the range of 5to 50% of the weight of the wet final particle. The silica material usedis for instance quartz, the latter being preferred at the moment. Theinvention is further related to the use of such beads in an expandedfluid bed system for separating various components from a liquidcontaining the desired component.

[0029] When preparing the agarose base matrix material which is the atpresent preferred bead matrix, an agarose solution containing the quartzparticles is emulgated in the presence of a thickening additive, asfurther exemplified below.

[0030] The emulsifying agent should be a low molecular weight surfaceactive compound and could be ionic or nonionic, for instance Span 20,Gafen LB-400 or another agent known in this art.

[0031] The thickening agent should be soluble in the dispersion mediumand could be selected among hydrophobic polysaccharide derivatives. Thepreferred agent at the moment is ethyl cellulose.

[0032] In order to further stabilize the beads the polymer chains in thebeads may preferably be crosslinked in a manner known per se. In case apolysaccharide like agarose is used the beads could for instance besuspended in a solution containing a crosslinking agent like abisepoxide or a halohydrin under conditions to give crosslinking ofadjacent chains in the agarose matrix (Porath (1971)).

[0033] Agarose beads, as well as crosslinked agarose beads, have beenused in a great number of chromatographic applications and there is anextensive literature describing various methods for derivatizing thebeads for use in ion exchange chromatography, affinity chromatography,etc. These methods are obvious choices for preparing supports forspecific applications. One such example is binding of protein A orprotein G to the agarose matrix by the cyanogen bromide method forpreparing a support for purification of IgG.

[0034] The invention is in another aspect related to a method forseparating sample components by the use of a stabilized fluidized bed.

[0035] The method comprises the steps of

[0036] loading a column for fluidized liquid bed processes with beadparticles derivatized to bind a desired component,

[0037] conditioning the particles with a solution under conditions sothat a stabilized fluidized bed is created,

[0038] feeding the so stabilized fluidized bed with a sample solutioncontaining the desired component through a bottom port of the column,whereby the desired component is bound to the beads and impurities ofthe sample solution are discharged through the top port of the column,

[0039] washing the column with a solution introduced through said bottomport under flow conditions maintaining the expanded bed, wherebyremaining unbound components of the sample solution are dischargedthrough the top port of the column,

[0040] introducing an elution solution into the column for releasing thebound sample constituents from the beads and collecting them, optionallyby using a fraction collector.

[0041] In one embodiment of the invention the elution step is carriedout by introducing the elution solution through the bottom port Go thecolumn and maintaining the bed in an expanded bed mode.

[0042] In another embodiment the gel bed is packed by applying an inletflow of an appropriate conditioning buffer through the upper port of thecolumn, and by moving down an upper adapter to the gel surface. Thisbuffer should of course not be allowed to release the desired componentfrom the beads. The elution solution is then introduced through theupper port of the column, the sample constituents are released from thebeads and finally collected in the outflow from the bottom port of thecolumn.

[0043] The advantages of using this method are readily appreciated incases when one or more components from cell culture experiments are tobe separated from other constituents of the medium. Solid cellparticles, which would cause clogging of a packed bed can easily beremoved from the system during the fluidized bed phase of the procedureand will not at all interfere.

[0044] The invention will now be illustrated by a series of examples.

EXAMPLE 1 Synthesis of Crosslinked Agarose Beads Containing QuartzParticles 10 g/100 ml. 6% Agarose

[0045] Bead Formation

[0046] An agarose solution was prepared by stirring 57 g of agarose in900 ml water at 90° C., until the agarose was dissolved and theviscosity of the solution was in the range of 250-700 cPs at 85° C.

[0047] To the stirred agarose solution 90 g of quartz particles (10-70μm, NGQ 200, Ernströms, Sweden) was added. The slurry was poured into asolution of 45 g ethyl cellulose (N-50, Hercules, USA) in 1000 ml oftoluene, at 60° C., in a cylindrical vessel equipped with a stirrer at100-200 rpm. Approximately 20 ml of Gafen LB 400 (Gaf,USA) in toluene(1:2, w/v) was gradually added to give the desired bead size. Thestirred suspension was cooled to room temperature and 1000 ml water wasadded. The mixture was left over night and the upper liquid phase wasremoved. The beads were washed with toluene, with water and finally wetsieved to yield a size distribution of 125-315 μm.

[0048] Crosslinking

[0049] Crosslinking with epichlorohydrin was essentially performed asdescribed by Porath et al (1971).

EXAMPLE 2 Synthesis of Agarose Beads Containing Quartz Particles. 13g/100 ml 4% Agarose

[0050] To a stirred agarose solution, prepared as in Example 1 but with12 g of agarose in 300 ml water, 40 g quartz (10-70 μm, NGQ 200,Ernströms, Sweden) was added. The slurry was suspended in a stirredsolution of 25 g ethyl cellulose (N-50, Hercules, USA) in 350 mltoluene, at 60° C. After one hour the suspension was allowed to reachroom temperature. The quartz containing agarose beads were washed, wetsieved and crosslinked as above.

EXAMPLE 3 Synthesis of Agarose Beads Containing Quartz Particles. 17g/100 ml, 6% Agarose

[0051] A slurry of quartz in agarose solution was prepared as in Example2, but with 28 g agarose in 450 ml water and with 75 g quartz. Thesuspension was suspended in a solution of 500 ml toluene and 50 g ethylcellulose and stirred for 3 hours at 60° C. After cooling to roomtemperature, the beads were washed, wet sieved and crosslinked as above.

EXAMPLE 4 Synthesis of Agarose Beads Containing Quartz Particles. 33g/100 ml, 6% Agarose

[0052] Prepared as in Example 3 but with 150 g quartz particles.

EXAMPLE 5 Synthesis of Crosslinked Dextran Beads Containing QuartzParticles

[0053] To a stirred solution of 16 g dextran (Mw 250 000), 3,5 g sodiumboro hydride and 1,6 g sodium hydroxide in 40 ml water, 3 g quartzparticles (10-45 μm, NGQ 325, Ernströms, Sweden) were added. The slurrywas added to a stirred solution of 3 g Gafen LB 400 and 3 g ethylhydroxyethyl cellulose (EHEC-XH, Hercules) in 75 m toluene in a roundbottom flask equipped with a blade stirrer at 500 rpm. The stirredsuspension was heated to 50° C. and 8 ml epichlorohydrin was added. Thesuspension was kept at 50° C. over night. The quartz containing beadswere then washed with ethanol and finally with slightly acidic water andwet sieved as above.

EXAMPLE 6 Synthesis of Porous Divinyl Benzene Beads, Containing QuartzParticles

[0054] Silanization of Quartz Particles

[0055] A slurry of 40 g quartz particles (10-70 μm, NGQ 200. Ernströms,Sweden) in 200 ml, 1 M sodium hydroxide was heated at 90° C. for 3hours. The cooled slurry was filtered and the quartz was washed withwater and dried. The quartz particles were transferred to 140 ml tolueneand 6,25 g imidazol was added. The stirred slurry was heated to 100° C.and 16,6 g dimethyloctadecylchlorosilane was added. The slurry wasmaintained at 100° C. over night and then cooled and filtered. Thesilanized quartz particles were washed with acetone, water and finallyacetone and dried at room temperature.

[0056] Suspension Polymerisation

[0057] A solution of 5 g polyvinyl alcohol (Mowiol 40-88, USA) in 100 mlwater was mixed with a solution of 15 g dextran (Mw 40 000) in 150 mlwater. To the stirred solution a slurry on 0,5 g benzoyl peroxide, 25 mldivinylbenzene, 25 ml 4-methyl-2-pentanol and 4 g silanized quartz wasslowly added and kept at 75° C. for 6 hours. The cooled suspension wasfiltered and the quartz containing divinyl benzene beads were washedwith alcohol and finally with water.

EXAMPLE 7 Sedimentation of Agarose Particles with and Without QuartzParticles

[0058] In this experiment various gels were loaded on a column suitablefor fluidized bed experiments and the height of the gel bed was measuredat various flow rates.

[0059] Gel 1: (125-280 μm) is an agarose gel synthesized as in Example 1but without addition of quartz. It has further been derivatized tocontain sulphonate groups which however is of no importance in theactual experiments.

[0060] Gel 2: (122-250 μm) is a quartz-containing agarose gelsynthesized as in Example 1. The quartz contents was 17 g of quartz per100 ml of agarose and it was further derivatized to containdiethylaminoethyl (DEAE) groups.

[0061] Gel 3: (125-280 μm) is a gel containing 33 g of quartz per 100 mlof agarose and was synthesized as Gel 2.

[0062] Each gel was loaded on a column (50×600 mm) and the height of thebed was measured. A flow of 10 ml/ml was applied and the height of thenow expanded bed was measured after it had stabilized. The flow wasincreased in steps of 10 ml/min and the subsequent bed height wasmeasured. The results showed that it was possible to apply considerablyhigher flow velocities using the beads of the invention and stillworking under stabilized fluidized bed conditions.

EXAMPLE 8 Purification of a Recombinant Anticoagulant Protein from an E.Coli Homogenate

[0063] An E.coli strain expressing the anticoagulant protein annexin Vwas grown in a complex nutrient medium in a fermentor. After harvest thecells were pelleted, resuspended in water and passed three times througha high pressure homogenizer. Triton X-100 was added to 0.5% finalconcentration and the pH was adjusted to 5.5 using acetic acid.

[0064] An expanded bed column (50 mm diameter, 60 cm high) was filled to11.5 cm (225 ml) with quartz containing agarose beads, according to thepresent invention, which were charged with diethylaminoethyl (DEAE)groups. The gel bed was expanded to 30 cm at 300 cm/h linear flow ratein equilibration buffer; 30 mM ammonium acetates pH 5.5. At the sameflow rate, 1600 ml of the E. coli homogenate was applied. The gel bedwas then washed with equilibration buffer and equilibration buffercontaining 80 mM NaCl, at 400 cm/h. The flow rate was then decreased to200 cm/h and the flow direction reversed in order to pack the gel. Theupper adapter was moved down to the bed surface and bound proteins wereeluted with equilibration buffer containing 250 mM NaCl at 100 cm/h.According to anticoagulant activity assays 80% of the applied annexin Vwas bound and eluted from the gel.

[0065] This purification was also successfully repeated in a pilot scaleexpanded bed column (200 m diameter, 95 cm high) containing 3.1 litergel. The sample volume was 8 liter.

EXAMPLE 9 Characterization of the Fluidized Bed Stability

[0066] An expanded bed column (50 mm diameter, 60 cm high) containing adistributor with a hole area of about 3% of the total area, was filledto 6.0 cm (118 ml) with quartz containing agarose beads with a particledistribution in the range of from 100 to 270 μm. The gel was expanded to30 cm at 300 cm/h linear flow rate in distilled water. The pressure dropover the distributor was 66% of the pressure drop in the bed.

[0067] A pulse input of 32 ml acetone solution with a concentration of0.25% was injected into the column as a tracer in a “stimulus responseexperiment” (see Levenspiel (1972)). The result expressed as the “vesseldispersion number” defined from the model “Dispersion model for smallextents of dispersion” was 19×10⁻³ (for definitions see Levenspiel(1972))

[0068] References

[0069] Buijs et al (1980) “Batch fluidized ion exchange column forstreams containing suspended particles, Journal of Chromatography 201,page 319-327.

[0070] Lydersen (1979), Fluid Flow and Heat Transfer, John Wiley & SonsLtd, Chichester, page 134-136.

[0071] Burns et al (1985:1) “Continuous Affinity Chromatography using aMagnetically Stabilized Fluidized Bed”, Biotechnology Progress 1(2) page95-103.

[0072] Burns et al (1985:2) “Dried Calcium Alginate/Magnetite Spheres: ANew Support for Chromatographic Separations and Enzyme Immobilization”Biotechnology and Bioengineering 27, page 137-145.

[0073] Porath et al (1971) “Agar derivatives for chromatogrphy,electrophoresis and gel bound enzymes. Desulphated and reducedcrosslinked agar and agarose in spherical bead form”, Journal ofChromatography 60, page 167-177.

[0074] Levenspiel (1972) “Chemical Reaction Engineering”, SecondEdition. John Wiley & Sons.

1. A method for recovering a desired component from a sample solutioncomprising the steps of (i) feeding the sample solution comprising thedesired component to be recovered into a column containing beads havingaffinity for the component, through a bottom port equipped with a flowdistributor, under stabilized fluidized bed conditions, whereby unboundcomponents of the sample solution are discharged through the top port ofthe column (ii) washing the column with a solution introduced throughsaid bottom port under flow conditions, maintaining an expanded bed,whereby remaining solid components and other impurities of the samplesolution are discharged through the top port of the column, (iii)introducing an elution solution for releasing sample components bound tothe beads and collecting said components.
 2. A method according to claim1, wherein step (iii) is carried out by allowing the beads to settle,optionally by applying an upper adapter onto the bead surface andintroducing a solution through the top port of the column so that apacked bed is achieved and thereafter eluting the column by applyingthrough the top port an elution medium in which the component isreleased from the beads.
 3. A method according to any one of claims 1and 2, characterized in that the beads forming the fluidized bed arechromatographic support beads according to any one of claims 5-10.
 4. Amethod according to any one of claims 1-3, characterized in that thesample solution is a cell culture medium containing solid cellparticles.
 5. Support beads for use in downstream processing,characterized in that they comprises a polymer matrix into which glassor silica particles have been incorporated.
 6. Support beads accordingto claim 5, characterized in that the polymer is an, optionallycrosslinked, polysaccharide.
 7. Support beads according to claim 6,characterized in that the polymer is agarose.
 8. Support beads accordingto any one of claims 5-7 characterized by having a diameter of from 100to 1000 μm and a silica or glass contents of 5-50% (wet weight). 9.Support beads according to any of claims 5-8 characterized in that theglass or silica particles have a size distribution in the range of from1 to 100 μm.
 10. Support beads according to any one of claims 5-9characterized in that the silica particles used are quartz particles.